PEER-REVIEWED ARTICLE bioresources.com Contributions of Thermotolerant Bacteria to Organic Matter Degradation under a Hyperthermophilic Pretreatment Process during Chicken Manure Composting Yun Cao,a,b,c Lin Wang,a,d Yuting Qian,a Yueding Xu,a,b,c Huashan Wu,a,b,c Jing Zhang,a,b,c Hongying Huang,a,b,c,* and Zhizhou Chang a,b,c Composting technology comprising hyperthermophilic pretreatment (at ≥ 85 °C for 2 to 4 h, HTPRT) and aerobic composting was adopted to accelerate organic matter transformation and enhance nitrogen retention in chicken manure composting. The differences in physio-chemical parameters, successions, and metabolism functions of the bacterial community between HTPRT (85 °C, 4 h) and conventional composting (CK) were compared. The HTPRT composting system reached maturity 18 days in advance of CK. The HTPRT piles showed a lower maximum N loss (27.1% vs. 39.0%). The bacterial structure in the HTPRT system differed remarkably from that in CK. Ureibacillus (22.7%) and Ammoniibacillus (14.1%) were the most predominant species in the thermophilic phase of HTPRT pile, while the curing phase was dominated by Thermobifida (12.8%) and Saccharomonospora (11.8%). The authors’ results suggest that HTPRT improved the physical properties of the feedstock by reducing the bulk density, which favored microbiological activity, and thus improving composting efficiency. Keywords: Hyperthermophilic pretreatment; Animal manure waste; Nitrogen retention; Thermotolerant bacteria; Microbial community Contact information: a: Circular Agriculture Research Center, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R. China; b: Key Laboratory of Crop and Livestock Integrated Farming, Ministry of Agriculture, Nanjing 210014, P.R. China; c: Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing 210014, P.R. China; d: Nanjing Institute of Agricultural Sciences in Jiangsu Hilly Area, Nanjing 210046, P.R. China; * Corresponding author: [email protected] INTRODUCTION With the increase in the intensive livestock production, the quantity of poultry waste has dramatically increased in recent years (China Statistical Yearbook 2016; Zhang et al. 2016a). Composting is regarded as an effective technology for converting organic wastes, such as poultry manures, into usable organic fertilizers or a soil amendment. However, in some cases, the physio-chemical characteristics of organic wastes may make the wastes unsuitable for conventional composting. These properties may result in a lower temperature during composting and cause insufficient decomposition and poor sanitation of the composts. Moreover, the composting process can involve a considerable generation of malodourous gases such as ammonia (NH3), hydrogen sulfide, and volatile organic compounds (Jurado et al. 2015). It is estimated that ammonia emissions contributed to 71% to 88% of nitrogen loss in composting facilities (Steiner et al. 2010). Cao et al. (2019). “Bacteria degradation: composting,” BioResources 14(3), 6747-6766. 6747 PEER-REVIEWED ARTICLE bioresources.com Composting is a self-heating aerobic process driven by the actions of microbial communities (Gannes et al. 2013a). These communities are affected by the types of substrates present and environmental conditions, such as temperature, ventilation conditions, and pH (Tortosa et al. 2017). The process of composting is generally comprised of three predictable phases, mesophilic, thermophilic, and curing (Gannes et al. 2013a; Zhang and Sun 2014). These temperature phases reflect the activities of successive microbial populations responsible for the degradation of organic matter. As a result, temperature is regarded as one of the crucial variables during transformation of organic matter (OM). To promote the efficient OM degradation, the composting process is expected to continuously remain in the thermophilic phase. Thus, various countermeasures have been used to increase the temperatures during the composting of animal manures: aeration control, bulk agent amendment (Jurado et al. 2015; Zhou et al. 2015; Zhao et al. 2017; Liu et al. 2019), and direct microbial inoculation (Zhao et al. 2017; 2018). In fact, compost conditions, such as the pH, C/N ratio, and oxygen concentration improvement, have significant influence on the growth of microbial communities (Zhang and Sun 2014; Tortosa et al. 2017). Recently, composting of organic wastes by using a hyperthermophilic pretreatment (HTPRT) process has been developed by Yamada et al. (2007) to solve malodorous odor problems related to ammonia emission during the composting process. During the process, feedstocks first were subjected to a HTPRT reactor followed by a conventional composting system. The temperature of the raw materials in the HTPRT reactor reached 100 ℃, and the maximum temperature was kept for at least 30 min. The HTPRT has been reported as a novel technology that has prominent features, such as high bioconversion efficiency, accelerated formation of humic acids, low ammonia emission, and accelerated removal of antibiotic resistance genes and mobile genetic elements during composting (Liao et al. 2018). Although there have been some reports concerning composting combined with an HTPRT process, the mechanism by which the HTPRT process stabilizes animal manures in the subsequent aerobic composting system remains to be further researched. Previous studies mainly have focused on the dynamic changes in the composition of the microbial communities responsible for OM conversion during different composting phases. Few studies have concerned the metabolic activity and functional diversity of the bacterial community, which are of great importance to reveal OM transformation in composting. The objectives of the study were to: 1) compare the difference in the succession and metabolism function of bacterial community between the HTPRT and conventional composting; 2) evaluate the effect of the HTPRT on organic C degradation during poultry manure composting; and 3) identify the relationship between the environmental factors, including temperature, moisture, ash, total organic carbon (TOC), total nitrogen (TN), + - ammonium (NH4 -N), and nitrate (NO3 -N) contents, and bacterial composition in the HTPRT composting process. EXPERIMENTAL Materials and HTPRT Process The raw materials of chicken manure and rice straw were obtained from Luhe Animal Experimental Base of Jiangsu Academy of Agricultural Sciences (Jiangsu, China). The rice straw was cut into small pieces ranging from 2 cm to 4 cm. The chemical Cao et al. (2019). “Bacteria degradation: composting,” BioResources 14(3), 6747-6766. 6748 PEER-REVIEWED ARTICLE bioresources.com parameters of the feedstocks before and after HTPRT are shown in Table 1. In order to match what is being done in a composting facility, the HTPRT process and the subsequent thermophilic composting process were conducted in separated systems. Details of the HTPRT process have previously been described in Cao et al. (2018). Briefly, in the HTPRT reactor (400 L, Fig. 1A), about 250 kg of the mixed chicken manure and rice straw were stirred using a blender inside the reactor. The mixed materials were heated by dimethyl silicone oil circulating until the preset HTPRT condition (85 °C) was reached and sustained for 4 h. Compulsory ventilation was set at the rate of 100 L/kg TS • h L-1 with a blower. After the temperature of the mixed material declined to ambient temperature, it was transferred to a vessel for the subsequent composting (Fig. 1B). Table 1. Properties of Raw Materials for Composting Materials Moisture (%) Total Organic Total Nitrogen C/N Carbon (g/kg) (g/kg) Chicken manure 71.2 288 25.62 11.24 Rice straw 9.5 405 6.12 66.17 A B Feedstock Hyperthermophilic Subsequent mixing pretreatment composting Fig. 1. Schematic diagram of the HTPRT reactor (A) and the HTPRT composting process (B) Composting setup The chicken manure and rice straw residues, with or without HTPRT, were adjusted to the same moisture content (62%), and then they were spread on separate plastic sheets and moved into stainless cubic bins (65 cm × 65 cm × 65 cm) with a volume of 300 L for the subsequent aerobic in-vessel composting. The bins were externally insulated with two layers of foam (5 cm in thickness) and aluminum foil thermal insulators to minimize the convective heat loss. A removable foam lid was put on the top of each reactor to facilitate intermittent mixing and sampling of the substrate during composting. A foam board was Cao et al. (2019). “Bacteria degradation: composting,” BioResources 14(3), 6747-6766. 6749 PEER-REVIEWED ARTICLE bioresources.com fixed on the inner side at the bottom. The uniformly distributed holes (2 cm in diameter) were cut both at the top and the bottom of the bin. The area of the holes accounted for approximately 1/4 of the total area of the foam board. Three thermocouples connected to a data logger were fitted at 15 cm, 30 cm, and 45 cm from the bottom of each bin to record the temperature of the composts at the interval of 60 min. A metal grid with 1 × 1 mm2 holes was fixed to sustain the composting materials and to allow aeration from the bottom. Fresh air was naturally supplied without forced aeration by turning the composting material manually at 1-week intervals throughout the 62-day-long composting process. While the substrate was being loaded into each bin, original samples were withdrawn (bottom, middle, and top) immediately for subsequent analyses, and the composting time was noted as day zero (Day